Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R

Abstract

In previous work, random genome deletion mutants of Corynebacterium glutamicum R were generated using the insertion sequence (IS) element IS31831 and the Cre/loxP excision system. One of these mutants, C. glutamicum strain RD41, resulting from the deletion of a 10.1-kb genomic region (⌬cgR_1595 through cgR_ 1604) from the WT strain, showed cell elongation, and several lines appeared on the cell surface (bamboo shape). The morphological changes were suppressed by overexpression of cgR_1596. Single disruption of cgR_ 1596 in WT C. glutamicum R resulted in morphological changes similar to those observed in the RD41 strain. CgR_1596 has a predicted secretion signal peptide in the amino-terminal region and a predicted NlpC/P60 domain, which is conserved in cell wall hydrolases, in the carboxyl-terminal region. In C. glutamicum R, CgR_ 0802, CgR_1596, CgR_2069, and CgR_2070 have the NlpC/P60 domain; however, only simultaneous disruption of cgR_1596 and cgR_2070, and not cgR_2070 single disruption, resulted in cell growth delay and more severe morphological changes than disruption of cgR_1596. Transmission electron microscopy revealed multiple septa within individual cells of cgR_1596 single and cgR_1596-cgR_2070 double disruptants. Taken together, these results suggest that cgR_1596 and cgR_2070 are involved in cell separation and cell growth in C. glutamicum.The gram-positive, high-GC-content bacterium Corynebacterium glutamicum has been one of the most important bacteria in industry for several decades due to its high production of amino acids such as glutamate (16). The bacterium often shows a V-shaped cell form under the microscope, and its coryneform rod-shaped cells have one side of their poles slightly wider than the other. Since its unique characteristics enable it to produce such large amounts of amino acids, most C. glutamicum research has remained focused on the creation of highly productive strains on the one hand and analysis of metabolic flow regulation on the other (14,15,32). Consequently, the unusual behavior of the bacterium to produce V-shaped cells has elicited less research attention. The cell division system resulting in V-shaped cells is known as snapping division (37). Besides C. glutamicum, other Actinomycetales such as Arthorobacter, Nocardia, and Mycobacteria are known to produce V-shaped cells (9,18,28,38). Although several genes involved in cell division (13,17,20,30,34,47) and cell morphology (21,35,44,45) in C. glutamicum have been characterized, the molecular mechanism of the snapping division is still largely unknown.Cell division is achieved by the consecutive actions of cell extension, chromosome replication and segregation, and cell separation. In most prokaryotic and eukaryotic species, cell separation starts by constriction and the subsequent formation of two equivalent daughter cells. After completion of chromosome replication and segregation of the daughter chromosomes to the two halves of the cell, constriction occurs at a predetermined site and two progeny cells are produced. ...

Genome-Wide Identification of In Vivo Binding Sites of GlxR, a Cyclic AMP Receptor Protein-Type Regulator in Corynebacterium glutamicum

“…3). C. glutamicum possesses at least two cell wall hydrolases (cgR_1596 and cgR_2070) involved in separation of two daughter cells during cell division (82) plays a minor or supportive role in cell separation in addition to the cgR_1596 protein (82). In silico analyses detected the GlxR-binding site upstream of cgR_2070 (43), and the site is highly similar to that in the upstream region of cgR_1596 (see Table S2 in the supplemental material).…”

Identification and expression analysis of a gene encoding a shikimate transporter of Corynebacterium glutamicum

“…C. glutamicum cells transformed with pCRF220 or pCRF221 were placed on a 1 % agar-padded slide containing BT medium with 2 % glucose as described previously (Tsuge et al, 2008). Images were acquired with an upright microscope (Olympus BX51) equipped with a 6100 objective (UPlanSApo, NA 1.4, oil immersion) and a CCD camera (DP70; Olympus).…”

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

“…12 The NlpC/P60 domain typically has hydrolytic activity and enzymes containing this domain include acyltransferases, amidases, and endopeptidases. 11 NlpC/P60 proteins have also been localized at the septa of dividing bacteria and mutations are phenotypically manifested by septation defects, 14,15 suggesting a role in separation of daughter cells during cell division or general maintenance of the cell wall. 16 Two genes in Mycobacterium marinum, a fish pathogen, which encode NlpC/ P60 domain-containing proteins are required for infection in zebra fish and cord formation seen in virulent mycobacteria, 13 thus adding to the diverse functions associated with proteins containing this domain.…”

“…17 This is the same number of NlpC/P60 proteins annotated in M. tuberculosis 12 and Corynebacterium glutamicum genomes. 15 MAP_1203 shows increased expression, nearly 25-fold, when MAP is exposed to milk, 18 a hyperosmolar environment that triggers a more invasive form of the bacterium. Although both MAP_1204 and MAP_1272c proteins are immunogenic, MAP_1272c in particular has been observed as a strong antigen in multiple studies.…”